Expression system

ABSTRACT

An immunogenic reagent which produces an immune response which is protective against  Bacillus anthracis,  said reagent comprising one or more polypeptides which together represent up to three domains of the full length Protective Antigen (PA) of  B. anthracis  or variants of these, and at least one of said domains comprises domain 1 or domain 4 of PA or a variant thereof. The polypeptides of the immunogenic reagent as well as full length PA are produced by expression from  E. coli.  High yields of polypeptide are obtained using this method. Cells, vectors and nucleic acids used in the method are also described and claimed.

This application claims priority to Great Britain Application No.0016702.3 filed on Jul. 8, 2000 and International Application No.PCT/GB01/03065 filed on Jul. 6, 2001 and published in English asInternational Publication No. WO 02/04646 A1 on Jan. 17, 2002, theentire contents of which are hereby incorporated by reference.

The present invention relates to polypeptides which produce an immuneresponse which is protective against infection by Bacillus anthracis, tomethods of producing these, to recombinant Escherichia coli cells,useful in the methods, and to nucleic acids and transformation vectorsused.

Present systems for expressing Protective Antigen (PA) for vaccinesystems use protease deficient Bacillus subtilis as the expression host.Although such systems are acceptable in terms of product quantity andpurity, there are significant drawbacks. Firstly, regulatory authoritiesare generally unfamiliar with this host, and licensing decisions may bedelayed as a result. More importantly, the currently used strains ofBacillus subtilis produce thermostable spores which require the use of adedicated production plant.

WO00/02522 describes in particular VEE virus replicons which express PAor certain immunogenic fragments.

E. coli is well known as an expression system for a range of humanvaccines. While the ability to readily ferment E. coli to very highcellular densities makes this bacterium an ideal host for the expressionof many proteins, previous attempts to express and purify recombinant PAfrom E. coli cytosol have been hindered by low protein yields andproteolytic degradation (Singh et al., J. Biol. Chem. (1989) 264;11099-11102, Vodkin et al., Cell (1993) 34; 693-697 and Sharma et al.,Protein Expr. purif. (1996), 7, 33-38).

A strategy for overexpressing PA as a stable, soluble protein in the E.coli cytosol has been described recently (Willhite et al., Protein andPeptide Letters, (1998), 5; 273-278). The strategy adopted is one ofadding an affinity tag sequence to the N terminus of PA, which allows asimple purification system. A problem with this system is that itrequires a further downstream processing step in order to remove the tagbefore the PA can be used.

Codon optimisation is a technique which is now well known and used inthe design of synthetic genes. There is a degree of redundancy in thegenetic code, in so far as most amino acids are coded for by more thanone codon sequence. Different organisms utilise one or other of thesedifferent codons preferentially. By optimising codons, it is generallyexpected that expression levels of the particular protein will beenhanced.

This is generally desirable, except where, as in the case of PA, higherexpression levels will result in proteolytic degradation and/or celltoxicity. In such cases, elevating expression levels might becounter-productive and result in significant cell toxicity.

Surprisingly however, the applicants have found that this is not thecase in E. coli and that in this system, codon optimisation results inexpression of unexpectedly high levels of recombinant PA, irrespectiveof the presence or absence of proteolytic enzymes within the strain.

Furthermore, it would appear that expression of a protective domain ofPA does not inhibit expression in E. coli.

The crystal structure of native PA has been elucidated (Petosa C., etal. Nature 385: 833-838,1997) and shows that PA consists of fourdistinct and functionally independent domains: domain 1, divided into1a, 1˜167 amino acids and 1b, 168-258 amino acids; domain 2, 259-487amino acids; domain 3, 488-595 amino acids and domain 4, 596-735 aminoacids.

The applicants have identified that certain domains appear to producesurprisingly good protective effects when used in isolation, in fusionproteins or in combination with each other.

According to the present invention there is provided an immunogenicreagent which produces an immune response which is protective againstBacillus anthracis, said reagent comprising one or more polypeptideswhich together represent up to three domains of the full lengthProtective Antigen (PA) of B. anthracis or variants of these, and atleast one of said domains comprises domain 1 or domain 4 of PA or avariant thereof.

Specifically, the reagent will comprise mixtures of polypeptides orfusion peptides wherein individual polypeptides comprise one of moreindividual domains of PA.

In particular, the reagent comprises polypeptide(s) comprising domain 1or domain 4 of PA or a variant thereof, in a form other than full lengthPA. Where present, domains are suitably complete, in particular domain 1is present in its entirety.

The term “polypeptide” used herein includes proteins and peptides.

As used herein, the expression “variant” refers to sequences of aminoacids which differ from the basic sequence in that one or more aminoacids within the sequence are deleted or substituted for other aminoacids, but which still produce an immune response which is protectiveagainst Bacillus anthracis. Amino acid substitutions may be regarded as“conservative” where an amino acid is replaced with a different aminoacid with broadly similar properties. Non-conservative substitutions arewhere amino acids are replaced with amino acids of a different type.Broadly speaking, fewer non-conservative substitutions will be possiblewithout altering the biological activity of the polypeptide. Suitablyvariants will be at least 60% identical, preferably at least 75%identical, and more preferably at least 90% identical to the PAsequence.

In particular, the identity of a particular variant sequence to the PAsequence may be assessed using the multiple alignment method describedby Lipman and Pearson, (Lipman, D. J. & Pearson, W. R. (1985) Rapid andSensitive Protein Similarity Searches, Science, vol 227, pp1435-1441).The “optimised” percentage score should be calculated with the followingparameters for the Lipman-Pearson algorithm:ktup=1, gap penalty=4 andgap penalty length=12. The sequences for which similarity is to beassessed should be used as the “test sequence” which means that the basesequence for the comparison, (SEQ ID NO 1), should be entered first intothe algorithm.

Preferably, the reagent of the invention includes a polypeptide whichhas the sequence of domain 1 and/or domain 4 of wild-type PA.

A particularly preferred embodiment of the invention comprises domain 4of the PA of B. anthracis.

These domains comprise the following sequences shown in the followingTable 1.

TABLE 1 Domain Amino acids of full-length PA* 4 596-735 1  1-258

These amino acid numbers refer to the sequence as shown in Welkos et al.Gene 69 (1988) 287-300 and are illustrated hereinafter as SEQ ID NOs 15(FIG. 4) and 3 (FIG. 3) respectively.

Domain 1 comprises two regions, designated 1a and 1b. Region 1acomprises amino acids 1-167 whereas region 1b is from amino acid168-258. It appears that region 1a is important for the production of agood protective response, and the full domain may be preferred.

In a particularly preferred embodiment, a combination of domains 1 and 4or protective regions thereof, are used as the immunogenic reagent whichgives rise to an immune response protective against B. anthracis. Thiscombination, for example as a fusion peptide, may be expressed using theexpression system of the invention as outlined hereinafter.

When domain 1 is employed, it is suitably fused to domain 2 of the PAsequence, and may preferably be fused to domain 2 and domain 3.

Such combinations and their use in prophylaxis or therapy forms afurther aspect of the invention.

Suitably the domains described above are part of a fusion protein,preferably with an N-terminal glutathione-s-transferase protein (GST).The GST not only assists in the purification of the protein, it may alsoprovide an adjuvant effect, possibly as a result of increasing the size.

The polypeptides of the invention are suitably prepared by conventionalmethods. For example, they may be synthesised or they may be preparedusing recombinant DNA technology. In particular, nucleic acids whichencode said domains are included in an expression vector, which is usedto transform a host cell. Culture of the host cell followed by isolationof the desired polypeptide can then be carried out using conventionalmethods. Nucleic acids, vectors and transformed cells used in thesemethods form a further aspect of the invention.

Generally speaking, the host cells used will be those that areconventionally used in the preparation of PA, such as Bacillus subtilis.

The applicants have found surprisingly that the domains either inisolation or in combination, may be successfully expressed in E. coliunder certain conditions.

Thus, the present invention further provides a method for producing animmunogenic polypeptide which produces an immune response which isprotective against B. anthracis, said method comprising transforming anE. coli host with a nucleic acid which encodes either (a) the protectiveantigen (PA) of Bacillus anthracis or a variant thereof which canproduce a protective immune response, or (b) a polypeptide comprising atleast one protective domain of the protective antigen (PA) of Bacillusanthracis or a variant thereof which can produce a protective immuneresponse as described above, culturing the transformed host andrecovering the polypeptide therefrom, provided that where thepolypeptide is the protective antigen (PA) of Bacillus anthracis or avariant thereof which can produce a protective immune response, thepercentage of guanidine and cytosine residues within the said nucleicacid is in excess of 35%.

Using these options, high yields of product can be obtained using afavoured expression host.

A table showing codons and the frequency with which they appear in thegenomes of Escherichia coli and Bacillus anthracis is shown in FIG. 1.It is clear that guanidine and cytosine appear much more frequently inE. coli than B. anthracis. Analysis of the codon usage content revealsthe following:

1^(st) letter of 2nd letter 3rd letter Total GC Species Codon GC ofCodon GC of Codon GC content E. coli 58.50% 40.70% 54.90% 51.37% B.anthracis 44.51% 31.07% 25.20% 33.59%

Thus it would appear that codons which are favoured by E. coli are thosewhich include guanidine or cytosine where possible.

By increasing the percentage of guanidine and cytosine nucleotides inthe sequence used to encode the immunogenic protein over that normallyfound in the wild-type B. anthracis gene, the codon usage will be suchthat expression in E. coli is improved.

Suitably the percentage of guanidine and cytosine residues within thecoding nucleic acid used in the invention, at least where thepolypeptide is the protective antigen (PA) of Bacillus anthracis or avariant thereof which can produce a protective immune response, is inexcess of 40%, preferably in excess of 45% and most preferably from50-52%.

High levels of expression of protective domains can be achieved, withusing the wild-type B. anthracis sequence encoding these units. However,the yields may be improved further by increasing the GC % of the nucleicacid as described above.

In a particular embodiment, the method involves the expression of PA ofB. anthracis.

Further according to the present invention, there is provided arecombinant Escherichia coli cell which has been transformed with anucleic acid which encodes the protective antigen (PA) of Bacillusanthracis or a variant thereof which can produce a protective immuneresponse, and wherein the percentage of guanidine and cytosine residueswithin the nucleic acid is in excess of 35%.

As before, suitably the percentage of guanidine and cytosine residueswithin the coding nucleic acid is in excess of 40%, preferably in excessof 45% and most preferably from 50-52%.

Suitably, the nucleic acid used to transform the E. coli cells of theinvention is a synthetic gene. In particular, the nucleic acid is of SEQID NO 1 as shown in FIG. 2 or a modified form thereof.

The expression “modified form” refers to other nucleic acid sequenceswhich encode PA or fragments or variants thereof which produce aprotective immune response but which utilise some different codons,provided the requirement for the percentage GC content in accordancewith the invention is met. Suitable modified forms will be at least 80%similar, preferably 90% similar and most preferably at least 95% similarto SEQ ID NO 1. In particular, the nucleic acid comprises SEQ ID NO 1.

In an alternative embodiment, the invention provides a recombinantEscherichia coli cell which has been transformed with a nucleic acidwhich encodes a protective domain of the protective antigen (PA) ofBacillus anthracis or a variant thereof which can produce a protectiveimmune response.

Preferably, the nucleic acid encodes domain 1 or domain 4 of B.anthracis.

Further according to the invention there is provided a method ofproducing an immunogenic polypeptide which produces an immune responsewhich is protective against B. anthracis, said method comprisingculturing a cell as described above and recovering the desiredpolypeptide from the culture. Such methods are well known in the art.

In yet a further aspect, the invention provides an E. colitransformation vector comprising a nucleic acid which encodes theprotective antigen (PA) of Bacillus anthracis or a variant thereof whichcan produce a protective immune response, and wherein the percentage ofguanidine and cytosine residues within the nucleic acid is in excess of35%.

A still further aspect of the invention comprises an E. colitransformation vector comprising a nucleic acid which encodes aprotective domain of the protective antigen (PA) of Bacillus anthracisor a variant thereof which can produce a protective immune response.

Suitable vectors for use in the transformation of E. coil are well knownin the art. For example, the T7 expression system provides goodexpression levels. However a particularly preferred vector comprisespAG163 obtainable from Avecia (UK).

A nucleic acid of SEQ ID NO 1 or a variant thereof which encodes PA andwhich has at least 35%, preferably at least 40%, more preferably atleast 45% and most preferably from 50-52% GC content form a furtheraspect of the invention.

If desired, PA of the variants, or domains can be expressed as a fusionto another protein, for example a protein which provides a differentimmunity, a protein which will assist in purification of the product ora highly expressed protein (e.g. thioredoxin, GST) to ensure goodinitiation of translation.

Optionally, additional systems will be added such as T7 lysozyme to theexpression system, to improve the repression of the system, although, inthe case of the invention, the problems associated with cell toxicityhave not been noted.

Any suitable E. coli strain can be employed in the process of theinvention. Strains which are deficient in a number of proteases (e.g.Ion⁻, ompT⁻) are available, which would be expected to minimiseproteolysis. However, the applicants have found that there is no need touse such strains to achieve good yields of product and that other knownstrains such as K12 produce surprisingly high product yields.

Fermentation of the strain is generally carried out under conventionalconditions as would be understood in the art. For example, fermentationscan be carried out as batch cultures, preferably in large shake flasks,using a complex medium containing antibiotics for plasmid maintenanceand with addition of IPTG for induction.

Suitably cultures are harvested and cells stored at −20° C. untilrequired for purification.

Suitable purification schemes for E. coli PA (or variant or domain)expression can be adapted from those used in B. subtilis expression. Theindividual purification steps to be used will depend on the physicalcharacteristics of recombinant PA. Typically an ion exchangechromatography separation is carried out under conditions which allowgreatest differential binding to the column followed by collection offractions from a shallow gradient. In some cases, a singlechromatographic step may be sufficient to obtain product of the desiredspecification.

Fractions can be analysed for the presence of the product using SDS PAGEor Western blotting as required.

As illustrated hereinafter, the successful cloning and expression of apanel of fusion proteins representing intact or partial domains of rPAhas been achieved. The immunogenicity and protective efficacy of thesefusion proteins against STI spore challenge has been assessed in the A/Jmouse model.

All the rPA domain proteins were immunogenic in A/J mice and conferredat least partial protection against challenge compared to the GSTcontrol immunised mice. The carrier protein, GST attached to theN-terminus of the domain proteins, did not impair the immunogenicity ofthe fusion proteins either in vivo, shown by the antibody responsestimulated in immunised animals, or in vitro as the fusion proteinscould be detected with anti-rPA antisera after Western blotting,indicating that the GST tag did not interfere with rPA epitoperecognition. Immunisation with the larger fusion proteins produced thehighest titres. In particular, mice immunised with the full length GST1-4 fusion protein produced a mean serum anti-rPA concentrationapproximately eight times that of the rPA immunised group (FIG. 5).Immunisation of mice with rPA domains 1-4 with the GST cleaved off,produced titres of approximately one half those produced by immunisationwith the fusion protein. Why this fusion protein should be much moreimmunogenic is unclear. It is possible that the increased size of thisprotein may have an adjuvantising effect on the immune effector cells.It did not stimulate this response to the same extent in the otherfusion proteins and any adjuvantising effect of the GST tag did notenhance protection against challenge as the cleaved proteins weresimilarly protective to their fusion protein counterparts.

Despite having good anti-rPA titres, some breakthrough in protection atthe lower challenge level of 10² MLD's, occurred in the groups immunisedwith GST1, cleaved 1, GST1b-2, GST1b-3 and GST1-3 and immunisation withthese proteins did not prolong the survival time of those mice that didsuccumb to challenge, compared with the GST control immunised mice. Thissuggests that the immune response had not been appropriately primed bythese proteins to achieve full resistance to the infection. As has beenshown in other studies in mice and guinea pigs (Little S. F. et al.1986. Infect. Immun. 52: 509-512, Turnbull P. C. B., et al., 1986.Infect. Immun. 52: 356-363) there is no precise correlation betweenantibody titre to PA and protection against challenge. However a certainthreshold of antibody is required for protection (Cohen S et. al., 2000Infect. Immun. 68: 4549-4558), suggesting that cell mediated componentsof the immune response are also required to be stimulated for protection(Williamson 1989).

GST1, GST1b-2 and GST1-2 were the least stable fusion proteins produced,as shown by SDS-Page and Western blotting results, possibly due to theproteins being more susceptible to degradation in the absence of domain3, and this instability may have resulted in the loss of protectiveepitopes.

The structural conformation of the proteins may also be important forstimulating a protective immune response. The removal of Domain 1a fromthe fusion proteins gave both reduced antibody titres and lessprotection against challenge, when compared to their intact counterpartsGST1-2 and GST1-3. Similarly, mice immunised with GST 1 alone werepartially protected against challenge, but when combined with domain 2,as the GST1-2 fusion protein, full protection was seen at the 10² MLDchallenge level. However the immune response stimulated by immunisationwith the GST1-2 fusion protein was insufficient to provide fullprotection against the higher 10³ MLD's challenge level, which againcould be due to the loss of protective epitopes due to degradation ofthe protein.

All groups immunised with truncates containing domain 4, including GST 4alone, cleaved 4 alone and a mixture of two individually expresseddomains, GST 1 and GST 4 were fully protected against challenge with 10³MLDs of STI spores (Table 1). Brossier et al showed a decrease inprotection in mice immunised with a mutated strain of B. anthracis thatexpressed PA without domain 4 (Brossier F., et al. 2000. Infect. Immun.68: 1781-1786) and this was confirmed in this study, where immunisationwith GST 1-3 resulted in breakthrough in protection despite goodantibody titres. These data indicate that domain 4 is the immunodominantsub-unit of PA. Domain 4 represents the 139 amino acids of the carboxyterminus of the PA polypeptide. It contains the host cell receptorbinding region (Little S. F. et al., 1996 Microbiology 142: 707-715),identified as being in and near a small loop located between amino acidresidues 679-693 (Varughese M., et al. 1999 Infect. Immun.67:1860-1865).

Therefore it is essential for host cell intoxication as it has beendemonstrated that forms of PA expressed containing mutations (Varughese1999 supra.) or deletions (Brossier 1999 supra.) in the region of domain4 are non-toxic. The crystal structure of PA shows domain 4, and inparticular a 19 amino acid loop of the domain (703-722), to be moreexposed than the other three domains which are closely associated witheach other (Petosa 1997 supra.). This structural arrangement may makedomain 4 the most prominent epitope for recognition by immune effectorcells, and therefore fusion proteins containing domain 4 would elicitthe most protective immune response.

This investigation has further elucidated the role of PA in thestimulation of a protective immune response demonstrating thatprotection against anthrax infection can be attributed to individualdomains of PA.

The invention will now be particularly described by way of example, withreference to the accompanying drawings in which:

FIG. 1 is a Table of codon frequencies found within E. coli and B.anthracis;

FIG. 2 shows the sequence of a nucleic acid according to the invention,which encodes PA of B. anthracis, as published by Welkos et al supra;and

FIG. 3 shows SEQ ID NOs 3-14, which are amino acid and DNA sequencesused to encode various domains or combinations of domains of PA asdetailed hereinafter;

FIG. 4 shows SEQ ID NOs 15-16 which are the amino acid and DNA sequencesof domain 4 of PA respectively; and

FIG. 5 is a table showing anti-rPA IgG concencentration, 37 days postprimary immunisation, from A/J mice immunised intramuscularly on days 1and 28 with 10 μg of fusion protein included PA fragment; results shownare mean±sem of samples taken from 5 mice per treatment group.

EXAMPLE 1

Investigation into Expression in E. coli

rPA expression plasmid pAG163: :rPA has been modified to substituteKm^(R) marker for original Tc^(R) gene. This plasmid has beentransformed into expression host E. coli BLR (DE3) and expression leveland solubility assessed. This strain is deficient in the intracellularprotease La (Ion gene product) and the outer membrane protease OmpT.

Expression studies did not however show any improvement in theaccumulation of soluble protein in this strain compared to Ion+K12 hoststrains (i.e. accumulation is prevented due to excessive proteolysis).It was concluded that any intracellular proteolysis of rPA was not dueto the action of La protease.

EXAMPLE 2

Fermentation Analysis

Further analysis of the fermentation that was done using the K12 strainUT5600 (DE3) pAG163: :rPA.

It was found that the rPA in this culture was divided between thesoluble and insoluble fractions (estimated 350 mg/L insoluble, 650 mg/Lfull length soluble). The conditions used (37° C., 1 mM IPTG forinduction) had not yielded any detectable soluble rPA in shake flaskcultures and given the results described in Example 1 above, thepresence of a large amount of soluble rPA is surprising. Nevertheless itappears that manipulation of the fermentation, induction and point ofharvest may allow stable accumulation of rPA in E. coli K12 expressionstrains.

EXAMPLE 3

A sample of rPA was produced from material initially isolated asinsoluble inclusion bodies from the UT5600 (DE3) pAG163: :rPAfermentation. Inclusion bodies were washed twice with 25 mM Tris-HCl pH8 and once with same buffer +2M urea. They were then solubilized inbuffer +8M urea and debris pelleted. Urea was removed by dilution into25 mM Tris-HCl pH 8 and static incubation overnight at 4° C. Dilutedsample was applied to Q sepharose column and protein eluted with NaClgradient. Fractions containing highest purity rPA were pooled, aliquotedand frozen at −70° C. Testing of this sample using 4-12% MES-SDS NuPAGEgel against a known standard indicated that it is high purity and low inendotoxin contamination.

EXAMPLE 4

Further Characterisation of the Product

N terminal sequencing of the product showed that the N-terminal sequenceconsisted of

-   -   MEVKQENRLL (SEQ ID NO 2)

This confirmed that the product was as expected with initiatormethionine left on.

The material was found to react in Western blot; MALDI -MS on the sampleindicated a mass of approx 82 700 (compared to expected mass of 82 915).Given the high molecular mass and distance from mass standard used (66KDa), this is considered an indication that material does not havesignificant truncation but does not rule out microheterogeneity withinthe sample.

EXAMPLE 5

Testing of Individual Domains of PA

Individual domains of PA were produced as recombinant proteins in E.coli as fusion proteins with the carrier proteinglutathione-s-transferase (GST), using the Pharmacia pGEX-6P-3expression system. The sequences of the various domains and the DNAsequence used to encode them are attached herewith as FIG. 3. Therespective amino acid and DNA sequences are provided in Table 2 below.

These fusion proteins were used to immunise A/J mice (Harlan Olac)intramuscularly with 10 μg of the respective fusion protein adsorbed to20% v/v alhydrogel in a total volume of 100 μl.

Animals were immunised on two occasions and their development ofprotective immunity was determined by challenge with spores of B.anthracis (STI strain) at the indicated dose levels. The table belowshows survivors at 14 days post-challenge.

Challenge level in spores/mouse Amino DNA acid SEQ SEQ ID Domains ID NONO 5 × 10⁴ 9 × 10⁴ 9 × 10⁵ 1 × 10⁶ 5 × 10⁶ GST-1 3 4 4/4 3/5 GST-1 + 2 56 4/4; 4/5; 5/5 5/5 GST-1b + 2 7 8 2/5 1/5 GST-1b + 2 + 3 9 10 2/5 3/5GST-1 + 2 + 3 11 12 Nd 4/5 3/5 GST-1 + 2 + 3 + 4 13 14 Nd 5/5 5/5 1 +2 + 3 + 4 13 14 Nd Nd 5/5 5/5

The data shows that a combination of all 4 domains of PA, whetherpresented as a fusion protein with GST or not, were protective up to ahigh challenge level. Removal of domain 4, leaving 1+2+3, resulted inbreakthrough at the highest challenge level tested, 9×10⁵. Domains 1+2were as protective as a combination of domains 1+2+3 at 9×10⁴ spores.However, removal of domain 1a to leave a GST fusion with domains 1b+2,resulted in breakthrough in protection at the highest challenge leveltested (9×10⁴) which was only slightly improved by adding domain 3.

The data indicates that the protective immunity induced by PA can beattributed to individual domains (intact domain 1 and domain 4) or tocombinations of domains taken as permutations from all 4 domains.

The amino acid sequence and a DNA coding sequence for domain 4 is shownin FIG. 4 as SEQ ID NOs 15 and 16 respectively.

EXAMPLE 6

Further Testing of Domains as Vaccines

DNA encoding the PA domains, amino acids 1-259, 168-488, 1-488,168-596,1-596, 260-735, 489-735, 597-735 and 1-735 (truncates GST1,GST1b-2, GST1-2, GST1b-3, GST1-3, GST2-4, GST3-4, GST4 and GST1-4respectively) were PCR amplified from B. anthracis Sterne DNA and clonedin to the XhoI/BamHI sites of the expression vector pGEX-6-P3(Amersham-Pharmacia) downstream and in frame of the lac promoter.Proteins produced using this system were expressed as fusion proteinswith an N-terminal glutathione-s-transferase protein (GST). Recombinantplasmid DNA harbouring the DNA encoding the PA domains was thentransformed in to E. coli BL21 for protein expression studies.

E. Coli BL21 harbouring recombinant pGEX-6-P3 plasmids were cultured inL-broth containing 50 μg/ml ampicillin, 30 μg/ml chloramphenicol and 1%w/v glucose. Cultures were incubated with shaking (170 rev min⁻¹) at 30°C. to an A_(600 nm) 0.4, prior to induction with 0.5 mM IPTG. Cultureswere incubated for a further 4 hours, followed by harvesting bycentrifugation at 10 000 rpm for 15 minutes.

Initial extraction of the PA truncates-fusion proteins indicated thatthey were produced as inclusion bodies. Cell pellets were resuspended inphosphate buffered saline (PBS) and sonicated 4×20 seconds in an icedwater bath. The suspension was centrifuged at 15 000 rpm for 15 minutesand cell pellets were then urea extracted, by suspension in 8M urea withstirring at room temperature for 1 hour. The suspension was centrifugedfor 15 minutes at 15000 rpm and the supernatant dialysed against 100 mMTris pH 8 containing 400 mM L-arginine and 0.1 mM EDTA, prior todialysis into PBS.

The successful refolding of the PA truncate-fusion proteins allowed themto be purified on a glutathione Sepharose CL-4B affinity column. Allextracts (with the exception of truncate GST1b-2, amino acid residues168-487) were applied to a 15 ml glutathione Sepharose CL-4B column(Amersham-Parmacia), previously equilibrated with PBS and incubated,with rolling, overnight at 4° C. The column was washed with PBS and thefusion protein eluted with 50 mM Tris pH 7, containing 150 mM NaCl, 1 mMEDTA and 20 mM reduced glutathione. Fractions containing the PAtruncates, identified by SDS-PAGE analysis, were pooled and dialysedagainst PBS. Protein concentration was determined using BCA (Perbio).

However truncate GST1b-2 could not be eluted from the glutathionesepharose CL-4B affinity column using reduced glutathione and wastherefore purified using ion exchange chromatography. Specifically,truncate GST1b-2 was dialysed against 20 mM Tris pH 8, prior to loadingonto a HiTrap Q column (Amersham-Parmacia), equilibrated with the samebuffer. Fusion protein was eluted with an increasing NaCl gradient of0-1M in 20 mM Tris pH8. Fractions containing the GST-protein werepooled, concentrated and loaded onto a HiLoad 26/60 Superdex 200 gelfiltration column (Amersham-Parmacia), previously equilibrated with PBS.Fractions containing fusion protein were pooled and the proteinconcentration determined by BCA (Perbio). Yields were between 1 and 43mg per litre of culture.

The molecular weight of the fragments and their recognition byantibodies to PA was confirmed using SDS PAGE and Western Blotting.Analysis of the rPA truncates by SDS Page and Western blotting showedprotein bands of the expected sizes. Some degradation in all of the rPAtruncates investigated was apparent showing similarity with recombinantPA expressed in B. subtilis. The rPA truncates GST1, GST1b-2 and GST1-2were particularly susceptible to degradation in the absence of domain 3.This has similarly been reported for rPA constructs containing mutationsin domain 3, that could not be purified from B. anthracis culturesupernatants (Brossier 1999), indicating that domain 3 may stabilisedomains 1 and 2.

Female, specific pathogen free A/J mice (Harlan UK) were used in thisstudy as these are a consistent model for anthrax infection (Welkos1986). Mice were age matched and seven weeks of age at the start of thestudy.

A/J mice were immunised on days 1 and 28 of the study with 10 μg offusion protein adsorbed to 20% of 1.3% v/v Alhydrogel (HCI Biosector,Denmark) in a total volume of 100 μl of PBS. Groups immunised with rPAfrom B. subtilis (Miller 1998), with recombinant GST control protein, orfusion proteins encoding domains 1, 4 and 1-4 which had the GST tagremoved, were also included. Immunising doses were administeredintramuscularly into two sites on the hind legs. Mice were blood sampled37 days post primary immunisation for serum antibody analysis by enzymelinked immunosorbant assay (ELISA).

Microtitre plates (Immulon 2, Dynex Technologies) were coated, overnightat 4° C. with 5 μg/ml rPA, expressed from B. subtilis (Miller 1998), inPBS except for two rows per plate which were coated with 5 μg/mlanti-mouse Fab (Sigma, Poole, Dorset). Plates were washed with PBScontaining 1% v/v Tween 20 (PBS-T) and blocked with 5% w/v skimmed milkpowder in PBS (blotto) for 2 hours at 37° C. Serum, double-diluted in 1%blotto, was added to the rPA coated wells and was assayed in duplicatetogether with murine IgG standard (Sigma) added to the anti-fab coatedwells and incubated overnight at 4° C. After washing, horse-radishperoxidase conjugated goat anti-mouse IgG (Southern BiotechnologyAssociates Inc.), diluted 1 in 2000 in PBS, was added to all wells, andincubated for 1 hour at 37° C. Plates were washed again before additionof the substrate 2,2′-Azinobis (3-ethylbenzthiazoline-sulfonic acid)(1.09 mM ABTS, Sigma). After 20 minutes incubation at room temperature,the absorbance of the wells at 414 nm was measured (Titertek Multiscan,ICN Flow). Standard curves were calculated using Titersoft version 3.1csoftware. Titres were presented as μg IgG per ml serum and groupmeans±standard error of the mean (sem) were calculated. The results areshown in FIG. 5.

All the rPA truncates produced were immunogenic and stimulated meanserum anti-rPA IgG concentrations in the A/J mice ranging from 6 μg perml, for the GST1b-2 truncate immunised group, to 1488 μg per ml, in theGST 1-4 truncate immunised group (FIG. 5). The GST control immunisedmice had no detectable antibodies to rPA.

Mice were challenged with B. anthracis STI spores on day 70 of theimmunisation regimen. Sufficient STI spores for the challenge wereremoved from stock, washed in sterile distilled water and resuspended inPBS to a concentration of 1×10⁷ and 1×10⁶ spores per ml. Mice werechallenged intraperitoneally with 0.1 ml volumes containing 1×10⁶ and1×10⁵ spores per mouse, respectively, and were monitored for 14 day postchallenge to determine their protected status. Humane end-points werestrictly observed so that any animal displaying a collection of clinicalsigns which together indicated it had a lethal infection, was culled.The numbers of immunised mice which survived 14 days post challenge areshown in Table 3.

TABLE 3 Challenge Level MLDs survivors/no. challenged (%) Domain 10²MLDs 10³ MLDs GST 1 3/5 (60) 1/5 (20)  GST 1b-2 1/5 (20) nd GST 1-2  5/5(100) 3/5 (60)  GST 1b-3 3/5 (60) nd GST 1-3 4/5 (80) nd GST 1-4 nd 5/5(100) GST 2-4 nd 5/5 (100) GST 3-4 nd 5/5 (100) GST 4  5/5 (100) 5/5(100) GST 1 + GST 4 nd 5/5 (100) Cleaved 1 1/5 (20) 2/5 Cleaved 4  5/5(100) 5/5 Cleaved 1-4 nd 5/5 rPA nd 4/4 (100) control 0/5 (0)  0/5 (0) 1 MLD = aprox. 1 × 10³ STI spores nd = not done

The groups challenged with 10³ MLD's of STI spores were all fullyprotected except for the GST1, GST1-2 and cleaved 1 immunised groups inwhich there was some breakthrough in protection, and the control groupimmunised with GST only, which all succumbed to infection with a meantime to death (MTTD) of 2.4±0.2 days. At the lower challenge level of10² MLD's the GST1-2, GST4 and cleaved 4—immunised groups were all fullyprotected, but there was some breakthrough in protection in the othergroups. The mice that died in these groups had a MTTD of 4.5±0.2 dayswhich was not significantly different from the GST control immunisedgroup which all died with a MTTD of 4±0.4 days.

1. An immunogenic reagent comprising one or more isolated polypeptides,wherein the one or more isolated polypeptides alone or together are nomore than three full domains of full length Protective Antigen (PA) ofBacillus anthracis; wherein the one or more isolated polypeptidescomprise at least one of domain 1, region 1b of domain 1, or domain 4 ofthe PA; and wherein the immunogenic reagent produces an immune responsethat is protective against B. anthracis.
 2. The immunogenic reagent ofclaim 1 which comprises domain 4 of the PA of B. anthracis.
 3. Theimmunogenic reagent of claim 1 which comprises domains 1 and
 4. 4. Theimmunogenic reagent of claim 1 wherein the domains are present in theform of a fusion polypeptide.
 5. The immunogenic reagent of claim 4which comprises domain 1 fused to domain
 2. 6. The immunogenic reagentof claim 5 further comprising domain 3 fused to domain 1 or domain
 2. 7.The immunogenic reagent of claim 1 wherein the polypeptide is fused to aglutathione-S-transferase (GST).
 8. An immunogenic reagent comprisingone or more isolated polypeptides, wherein the one or more isolatedpolypeptides alone or together are no more than three full domains offull length Protective Antigen (PA) of B. anthracis; wherein the one ormore isolated polypeptides comprise at least one of domain 1, region 1bof domain 1, or domain 4 of the PA; and wherein the immunogenic reagentproduces an immune response that is protective against B. anthracis; andwherein one or more of the isolated polypeptides are fused to aglutathione-S-transferase (GST).
 9. The immunogenic reagent of claim 8wherein the polypeptide fused to the GST is domain 1; domains 1 and 2;domains 1, 2, and 3; region 1b and domain 2; region 1b and 2 and 3;domains 1and 4; domain 4, domains 2, 3, and 4; or domains 3 and 4.